Treatment of stroke using placental stem cells

ABSTRACT

The present invention provides a method of extracting and recovering embryonic-like stem cells, including, but not limited to pluripotent or multipotent stem cells, from an exsanguinated human placenta. A placenta is treated to remove residual umbilical cord blood by perfusing an exsanguinated placenta, preferably with an anticoagulant solution, to flush out residual cells. The residual cells and perfusion liquid from the exsanguinated placenta are collected, and the embryonic-like stem cells are separated from the residual cells and perfusion liquid. The invention also provides a method of utilizing the isolated and perfused placenta as a bioreactor in which to propagate endogenous cells, including, but not limited to, embryonic-like stem cells. The invention also provides methods for propagation of exogenous cells in a placental bioreactor and collecting the propagated exogenous cells and bioactive molecules therefrom.

This application is a continuation of U.S. patent application Ser. No.12/259,259, filed Oct. 27, 2008, which is a continuation of U.S. patentapplication Ser. No. 10/874,828, filed Jun. 22, 2004, now U.S. Pat. No.7,468,276, which is a continuation of U.S. patent application Ser. No.10/076,180, filed Feb. 13, 2002, now abandoned, which claims benefit ofU.S. Provisional Application No. 60/268,560, filed Feb. 14, 2001, andwhich is a continuation-in-part of U.S. patent application Ser. No.10/004,942, filed Dec. 5, 2001, now U.S. Pat. No. 7,045,148, which claimbenefit of U.S. Provisional Application No. 60/251,900, filed Dec. 6,2000; and is a continuation of U.S. patent application Ser. No.10/874,828, filed Jun. 22, 2004, now U.S. Pat. No. 7,468,276; each ofwhich is hereby incorporated by reference herein in its entirety.

1. INTRODUCTION

The present invention relates to methods of exsanguinating and perfusinga placenta following expulsion from the uterus, e.g., after birth. Thepresent invention relates to methods of treating and culturing anisolated placenta for the propagation of embryonic-like stem cellsoriginating from the placenta and exogenous sources. The presentinvention further relates to the use of a cultured placenta as abioreactor to produce biological materials or culture cells, tissues andorganoids. The present invention also relates to stem cell collectionand propagation, and in particular, to the collection of embryonic-likestem cells and other multipotent stem cells from placentas. The presentinvention relates to embryonic-like stem cells originating from apost-partum placenta.

2. BACKGROUND OF THE INVENTION

There is considerable interest in the identification, isolation andgeneration of human stem cells. Human stem cells are totipotential orpluripotential precursor cells capable of generating a variety of maturehuman cell lineages. This ability serves as the basis for the cellulardifferentiation and specialization necessary for organ and tissuedevelopment.

Recent success at transplanting such stem cells have provided newclinical tools to reconstitute and/or supplement bone marrow aftermyeloablation due to disease, exposure to toxic chemical and/orradiation. Further evidence exists that demonstrates that stem cells canbe employed to repopulate many, if not all, tissues and restorephysiologic and anatomic functionality. The application of stem cells intissue engineering, gene therapy delivery and cell therapeutics is alsoadvancing rapidly.

Many different types of mammalian stem cells have been characterized.For example, embryonic stem cells, embryonic germ cells, adult stemcells or other committed stem cells or progenitor cells are known.Certain stem cells have not only been isolated and characterized buthave also been cultured under conditions to allow differentiation to alimited extent. A basic problem remains, however, in that obtainingsufficient quantities and populations of human stem cells which arecapable of differentiating into all cell types is near impossible. Stemcells are in critically short supply. These are important for thetreatment of a wide variety of disorders, including malignancies, inbornerrors of metabolism, hemoglobinopathies, and immunodeficiencies. Itwould be highly advantageous to have a source of more embryonic stemcells.

Obtaining sufficient numbers of human stem cells has been problematicfor several reasons. First, isolation of normally occurring populationsof stem cells in adult tissues has been technically difficult and costlydue, in part, to very limited quantity found in blood or tissue.Secondly, procurement of these cells from embryos or fetal tissue,including abortuses, has raised religious and ethical concerns. Thewidely held belief that the human embryo and fetus constituteindependent life has prompted governmental restrictions on the use ofsuch sources for all purposes, including medical research. Alternativesources that do not require the use of cells procured from embryonic orfetal tissue are therefore essential for further progress in the use ofstem cells clinically. There are, however, few viable alternativesources of stem cells, particularly human stem cells, and thus supply islimited. Furthermore, harvesting of stem cells from alternative sourcesin adequate amounts for therapeutic and research purposes is generallylaborious, involving, e.g., harvesting of cells or tissues from a donorsubject or patient, culturing and/or propagation of cells in vitro,dissection, etc.

For example, Caplan et al. (U.S. Pat. No. 5,486,359 entitled “Humanmesenchymal stem cells,” issued Jan. 23, 1996), discloses humanmesenchymal stem cell (hMSC) compositions derived from the bone marrowthat serve as the progenitors for mesenchymal cell lineages. Caplan etal. discloses that hMSCs are identified by specific cell surface markersthat are identified with monoclonal antibodies. Homogeneous hMSCcompositions are obtained by positive selection of adherent marrow orperiosteal cells that are free of markers associated with eitherhematopoietic cell or differentiated mesenchymal cells. These isolatedmesenchymal cell populations display epitopic characteristics associatedwith mesenchymal stem cells, have the ability to regenerate in culturewithout differentiating, and have the ability to differentiate intospecific mesenchymal lineages when either induced in vitro or placed invivo at the site of damaged tissue. The drawback of such methods,however, is that they require harvesting of marrow or periosteal cellsfrom a donor, from which the MSCs must be subsequently isolated.

Hu et al. (WO 00/73421 entitled “Methods of isolation, cryopreservation,and therapeutic use of human amniotic epithelial cells,” published Dec.7, 2000) discloses human amniotic epithelial cells derived from placentaat delivery that are isolated, cultured, cryopreserved for future use,or induced to differentiate. According to Hu et al., a placenta isharvested immediately after delivery and the amniotic membrane separatedfrom the chorion, e.g., by dissection. Amniotic epithelial cells areisolated from the amniotic membrane according to standard cell isolationtechniques. The disclosed cells can be cultured in various media,expanded in culture, cryopreserved, or induced to differentiate. Hu etal. discloses that amniotic epithelial cells are multipotential (andpossibly pluripotential), and can differentiate into epithelial tissuessuch as corneal surface epithelium or vaginal epithelium. The drawbackof such methods, however, is that they are labor-intensive and the yieldof stem cells is very low. For example, to obtain sufficient numbers ofstem cells for typical therapeutic or research purposes, amnioticepithelial cells must be first isolated from the amnion by dissectionand cell separation techniques, then cultured and expanded in vitro.

Umbilical cord blood (cord blood) is a known alternative source ofhematopoietic progenitor stem cells. Stem cells from cord blood areroutinely cryopreserved for use in hematopoietic reconstitution, awidely used therapeutic procedure used in bone marrow and other relatedtransplantations (see e.g., Boyse et al., U.S. Pat. No. 5,004,681,“Preservation of Fetal and Neonatal Hematopoietin Stem and ProgenitorCells of the Blood”, Boyse et al., U.S. Pat. No. 5,192,553, entitled“Isolation and preservation of fetal and neonatal hematopoietic stem andprogenitor cells of the blood and methods of therapeutic use”, issuedMar. 9, 1993). Conventional techniques for the collection of cord bloodare based on the use of a needle or cannula, which is used with the aidof gravity to drain cord blood from (i.e., exsanguinate) the placenta(Boyse et al., U.S. Pat. No. 5,192,553, issued Mar. 9, 1993; Boyse etal., U.S. Pat. No. 5,004,681, issued Apr. 2, 1991; Anderson, U.S. Pat.No. 5,372,581, entitled Method and apparatus for placental bloodcollection, issued Dec. 13, 1994; Hessel et al., U.S. Pat. No.5,415,665, entitled Umbilical cord clamping, cutting, and bloodcollecting device and method, issued May 16, 1995). The needle orcannula is usually placed in the umbilical vein and the placenta isgently massaged to aid in draining cord blood from the placenta.Thereafter, however, the drained placenta has been regarded as having nofurther use and has typically been discarded. A major limitation of stemcell procurement from cord blood, moreover, has been the frequentlyinadequate volume of cord blood obtained, resulting in insufficient cellnumbers to effectively reconstitute bone marrow after transplantation.

Naughton et al. (U.S. Pat. No. 5,962,325 entitled “Three-dimensionalstromal tissue cultures” issued Oct. 5, 1999) discloses that fetalcells, including fibroblast-like cells and chondrocyte-progenitors, maybe obtained from umbilical cord or placenta tissue or umbilical cordblood. Naughton et al. (U.S. Pat. No. 5,962,325) discloses that suchfetal stromal cells can be used to prepare a “generic” stromal orcartilaginous tissue. Naughton et al. also discloses that a “specific”stromal tissue may be prepared by inoculating a three-dimensional matrixwith fibroblasts derived from a particular individual who is later toreceive the cells and/or tissues grown in culture in accordance with thedisclosed methods. The drawback of such an approach however, is that itis labor intensive. According to the methods disclosed in Naughton etal., to recover fetal stromal cells from the umbilical cord or placentarequires dissection of these tissues, mincing of the tissue into piecesand disaggregation. Furthermore, to obtain adequate amounts of the fetalstromal cells from umbilical cord blood, as well as the umbilical cordand placenta, requires further expansion ex vivo.

Currently available methods for the ex vivo expansion of cellpopulations are also labor-intensive. For example, Emerson et al.(Emerson et al., U.S. Pat. No. 6,326,198 entitled “Methods andcompositions for the ex vivo replication of stem cells, for theoptimization of hematopoietic progenitor cell cultures, and forincreasing the metabolism, GM-CSF secretion and/or IL-6 secretion ofhuman stromal cells”, issued Dec. 4, 2001); discloses methods, andculture media conditions for ex vivo culturing of human stem celldivision and/or the optimization of human hematopoietic progenitor stemcells. According to the disclosed methods, human stem cells orprogenitor cells derived from bone marrow are cultured in a liquidculture medium that is replaced, preferably perfused, eithercontinuously or periodically, at a rate of 1 ml of medium per ml ofculture per about 24 to about 48 hour period. Metabolic products areremoved and depleted nutrients replenished while maintaining the cultureunder physiologically acceptable conditions.

Kraus et al. (Kraus et al., U.S. Pat. No. 6,338,942, entitled “Selectiveexpansion of target cell populations”, issued Jan. 15, 2002) disclosesthat a predetermined target population of cells may be selectivelyexpanded by introducing a starting sample of cells from cord blood orperipheral blood into a growth medium, causing cells of the target cellpopulation to divide, and contacting the cells in the growth medium witha selection element comprising binding molecules with specific affinity(such as a monoclonal antibody for CD34) for a predetermined populationof cells (such as CD34 cells), so as to select cells of thepredetermined target population from other cells in the growth medium.

Rodgers et al. (U.S. Pat. No. 6,335,195 entitled “Method for promotinghematopoietic and mesenchymal cell proliferation and differentiation,”issued Jan. 1, 2002) discloses methods for ex vivo culture ofhematopoietic and mesenchymal stem cells and the induction oflineage-specific cell proliferation and differentiation by growth in thepresence of angiotensinogen, angiotensin I (AI), AI analogues, AIfragments and analogues thereof, angiotensin II (AII), AII analogues,AII fragments or analogues thereof or AII AT₂ type 2 receptor agonists,either alone or in combination with other growth factors and cytokines.The stem cells are derived from bone marrow, peripheral blood orumbilical cord blood. The drawback of such methods, however, is thatsuch ex vivo methods for inducing proliferation and differentiation ofstem cells are time-consuming, as discussed above, and also result inlow yields of stem cells.

Naughton et al., (U.S. Pat. No. 6,022,743 entitled “Three-dimensionalculture of pancreatic parenchymal cells cultured living stromal tissueprepared in vitro,” issued Feb. 8, 2000) discloses a tissue culturesystem in which stem cells or progenitor cells (e.g., stromal cells suchas those derived from umbilical cord cells, placental cells, mesenchymalstem cells or fetal cells) are propagated on three-dimensional supportrather than as a two-dimensional monolayer in, e.g., a culture vesselsuch as a flask or dish.

Because of restrictions on the collection and use of stem cells, and theinadequate numbers of cells typically collected from cord blood, stemcells are in critically short supply. Stem cells have the potential tobe used in the treatment of a wide variety of disorders, includingmalignancies, inborn errors of metabolism, hemoglobinopathies, andimmunodeficiencies. There is a critical need for a readily accessiblesource of large numbers of human stem cells for a variety of therapeuticand other medically related purposes. The present invention addressesthat need and others.

3. SUMMARY OF THE INVENTION

The present invention relates to a mammalian placenta, preferably human,which following expulsion from the uterus has been treated and culturedto produce multipotent stem cells (e.g., committed progenitor cells),embryonic-like stem cells and other biological materials. In particular,the present invention provides methods of perfusing and exsanguinating aplacenta post birth. The present invention provides methods ofexsanguinating and perfusing a placenta under sterile conditions for aperiod of at least two to greater than forty-eight hours followingexpulsion of the placenta from the uterus. In a preferred embodiment,the placenta is perfused with a solution containing factors to enhancethe exsanguination, such as anticoagulant factors. In anotherembodiment, the placenta is perfused with a solution containing factorsto enhance the sterile conditions, such as antimicrobial and antiviralagents. In a preferred embodiment, the placenta is perfused with asolution containing growth factors. Such solutions which contains growthfactors and other culture components but without anticoagulants arereferred to as culture solution.

In another preferred embodiment of the invention, the placenta isperfused to remove blood, residual cells, proteins and any otherresidual material. The placenta may be further processed to removematerial debris. Perfusion is normally continued with an appropriateperfusate for at least two to more than twenty-four hours. In severaladditional embodiments, of the invention, the placenta is perfused forat least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours prior to thecollection of stem cells. The perfusate collected from any of these timepoints may also provide a source of embryonic-like stem cells. It shouldbe understood that the first collection of blood from the placenta isreferred to as cord blood which contains predominantly CD34+ and CD38+hematopoietic progenitor cells. Within the first twenty-four hours ofpost-partum perfusion, CD34+ and CD38− hematopoietic progenitor cellscan be isolated from the placenta along with CD34+ and CD38− cells.After about twenty-four hours of perfusion, CD34− and CD38− cells can beisolated from the placenta along with the aforementioned cells.

The present invention relates to an isolated placenta that has beenexsanguinated and perfused under sterile conditions. In a preferredembodiment, the invention provides an isolated placenta that has beenexsanguinated and perfused to remove all residual cells and cultured fora period of two to twenty four hours following expulsion from theuterus. The present invention also provides an isolated placenta thathas been treated and cultured to result in a viable organ capable ofproducing embryonic-like stem cells, progenitor cells and otherbiological materials.

The present invention relates to a stem cell producing apparatus whichcomprises a post-partum mammalian placenta which has been exsanguinatedand perfused, a means for incubating or culturing the placenta; and ameans for detecting stem cells. In another embodiment, the apparatus ofthe invention further comprises a collection device and/or a means forseparating the collected cells. In another embodiment, the apparatus ofthe invention further comprises a means for monitoring and adjusting theculture conditions and collection of cells.

The present invention also provides methods of incubating and culturingan isolated exsanguinated placenta under the appropriate conditions toallow for the production of embryonic-like stem cells that originatefrom the placenta. In accordance with the present invention,embryonic-like stem cells are obtained from a placenta followingexpulsion from the uterus. The placenta is exsanguinated and perfusedfor a period of at least two to twenty four hours to remove all residualcells. The exsanguinated placenta is then cultured under the appropriateconditions to allow for the production of endogenous stem cellsoriginating from the placenta, including, but not limited toembryonic-like stem cells, and pluripotent or multipotent stem cells. Ina preferred embodiment, the exsanguinated placenta is cultured in thepresence of growth factors, such as PDGF and EGF.

The present invention further provides methods of treating and culturingan isolated placenta for use as a bioreactor for the propagation ofendogenous stem cells originating from the placenta. The presentinvention provides methods of treating and culturing an isolatedplacenta for use as a bioreactor for the propagation of exogenous cellsand biological materials, e.g., antibodies, proteins, oligonucleotides,hormones, viruses, cytokines and enzymes. The present invention alsoprovides propagation and collection of embryonic-like stem cells andother pluripotent and multipotent stem cells from placentas. Thecultured placenta may be used repeatedly as a bioreactor and may becultured over a period of days, months and even years. The culturedplacenta may be maintained by periodically or continuously removing aportion of a culture medium or perfusion fluid that is introduced intothe system and from which the propagated cells or produced biologicalmaterials may be recovered, and replaced with fresh medium or perfusateliquid.

In another embodiment, the invention provides a method of utilizing theisolated and perfused placenta as a bioreactor in which to propagateendogenous cells, including, but not limited to, embryonic-like stemcells, progenitor cells, pluripotent cells and multipotent cells. Theendogenous cells propagated in the placental bioreactor may becollected, and/or bioactive molecules recovered from the perfusate,culture medium or from the placenta cells themselves.

In another embodiment, the invention provides a method of utilizing theisolated and perfused placenta as a bioreactor in which to propagateexogenous cells. In accordance with this embodiment, the inventionrelates to an isolated placenta which contains a cell not derived fromthe placenta, wherein the engraftment of said cell into the placenta maystimulate the placenta to produce embryonic-like stem cells or whereinthe engrafted cell produces signals, such a cytokines and growthfactors, which may stimulate the placenta to produce stem cells. Inaccordance with this embodiment, the placenta may be engrafted withcells not placental in origin obtained from the infant associated withthe placenta. In another embodiment, the placenta may be engrafted withcells not placental in origin obtained from the parents or siblings ofthe infant associated with the placenta. The exogenous cells propagatedin the placental bioreactor may be collected, and/or bioactive moleculesrecovered from the perfusate, culture medium or from the placenta cellsthemselves.

The present invention provides embryonic-like stem cells that originatefrom a placenta. The embryonic-like stem cells of the invention may becharacterized by measuring changes in morphology and cell surfacemarkers using techniques such as flow cytometry and immunocytochemistry,and measuring changes in gene expression using techniques, such as PCR.In one embodiment of the invention, such embryonic-like stem cells maybe characterized by the presence of the following cell surface markers:CD10+, CD29+, CD34−, CD38−, CD44+, CD45−, CD54+, CD90+, SH2+, SH3+,SH4+, SSEA3−, SSEA4−, OCT-4+, and ABC-p+. In a preferred embodiment,such embryonic-like stem cells may be characterized by the presence ofcell surface markers OCT-4+ and APC-p+. Embryonic-like stem cellsoriginating from placenta the have characteristics of embryonic stemcells but are not derived from the embryo. In other words, the inventionencompasses OCT-4+ and ABC-p+ cells that are undifferentiated stem cellsthat are isolated from post-partum perfused placenta. Such cells are asversatile (e.g., pluripotent) as human embryonic stem cells. Asmentioned above, a number of different pluripotent or multipotent stemcells can be isolated from the perfused placenta at different timepoints e.g., CD34+/CD38+, CD34+/CD38−, and CD34−/CD38−hematopoieticcells. According to the methods of the invention, human placenta is usedpost-birth as the source of embryonic-like stem cells.

In another embodiment, the invention provides a method for isolatingother embryonic-like and/or multipotent or pluripotent stem cells froman extractant or perfusate of a exsanguinated placenta.

The present invention relates to pharmaceutical compositions whichcomprise the embryonic-like stem cells of the invention. The presentinvention further relates to an isolated homogenous population of humanplacental stem cells which has the potential to differentiate into allcell types. The invention also encompasses pharmaceutical compositionshave high concentrations (or larger populations) of homogenoushematopoietic stem cells including but not limited to CD34+/CD38− cells;and CD34−/CD38− cells one or more of these cell populations can be usedwith or as a mixture with cord blood hematopoietic cells i.e.,CD34+/CD38+ hematopoietic cells for transplantation and other uses.

The stem cells obtained by the methods of the invention have a multitudeof uses in transplantation to treat or prevent disease. In oneembodiment of the invention, they are used to renovate and repopulatetissues and organs, thereby replacing or repairing diseased tissues,organs or portions thereof.

3.1. Definitions

As used herein, the term “bioreactor” refers to an ex vivo system forpropagating cells, producing or expressing biological materials andgrowing or culturing cells tissues, organoids, viruses, proteins,polynucleotides and microorganisms.

As used herein, the term “embryonic stem cell” refers to a cell that isderived from the inner cell mass of a blastocyst (e.g., a 4- to5-day-old human embryo) and that is pluripotent.

As used herein, the term “embryonic-like stem cell” refers to a cellthat is not derived from the inner cell mass of a blastocyst. As usedherein, an “embryonic-like stem cell” may also be referred to as a“placental stem cell.” An embryonic-like stem cell is preferablypluripotent. However, the stem cells which may be obtained from theplacenta include embryonic-like stem cells, multipotent cells, andcommitted progenitor cells. According to the methods of the invention,embryonic-like stem cells derived from the placenta may be collectedfrom the isolated placenta once it has been exsanguinated and perfusedfor a period of time sufficient to remove residual cells.

As used herein, the term “exsanguinated” or “exsanguination,” when usedwith respect to the placenta, refers to the removal and/or draining ofsubstantially all cord blood from the placenta. In accordance with thepresent invention, exsanguination of the placenta can be achieved by,for example, but not by way of limitation, draining, gravity inducedefflux, massaging, squeezing, pumping, etc. In a preferred embodiment,exsanguination of the placenta may further be achieved by perfusing,rinsing or flushing the placenta with a fluid that may or may notcontain agents, such as anticoagulants, to aid in the exsanguination ofthe placenta.

As used herein, the term “perfuse” or “perfusion” refers to the act ofpouring or passaging a fluid over or through an organ or tissue,preferably the passage of fluid through an organ or tissue withsufficient force or pressure to remove any residual cells, e.g.,non-attached cells from the organ or tissue. As used herein, the term“perfusate” refers to the fluid collected following its passage throughan organ or tissue. In a preferred embodiment, the perfusate containsone or more anticoagulants.

As used herein, the term “exogenous cell” refers to a “foreign” cell,i.e., a heterologous cell (i.e., a “non-self” cell derived from a sourceother than the placental donor) or autologous cell (i.e., a “self” cellderived from the placental donor) that is-derived from an organ ortissue other than the placenta.

As used herein, the term “organoid” refers to an aggregation of one ormore cell types assembled in superficial appearance or in actualstructure as any organ or gland of a mammalian body, preferably thehuman body.

As used herein, the term “multipotent cell” refers to a cell that hasthe capacity to grow into any of subset of the mammalian body'sapproximately 260 cell types. Unlike a pluripotent cell, a multipotentcell does not have the capacity to form all of the cell types.

As used herein, the term “pluripotent cell” refers to a cell that hascomplete differentiation versatility, i.e., the capacity to grow intoany of the mammalian body's approximately 260 cell types. A pluripotentcell can be self-renewing, and can remain dormant or quiescent within atissue. Unlike a totipotent cell (e.g., a fertilized, diploid egg cell),an embryonic stem cell cannot usually form a new blastocyst.

As used herein, the term “progenitor cell” refers to a cell that iscommitted to differentiate into a specific type of cell or to form aspecific type of tissue.

As used herein, the term “stem cell” refers to a master cell that canreproduce indefinitely to form the specialized cells of tissues andorgans. A stem cell is a developmentally pluripotent or multipotentcell. A stem cell can divide to produce two daughter stem cells, or onedaughter stem cell and one progenitor (“transit”) cell, which thenproliferates into the tissue's mature, fully formed cells.

As used herein, the term “totipotent cell” refers to a cell that is ableto form a complete embryo (e.g., a blastocyst).

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a cross-sectional view of the cannulation of the vein andartery of a placenta to perfuse the placenta and then collect theperfusate.

FIGS. 2 a-e are schematics showing the collection, clamping, perfusion,collection and storage of an exsanguinated and perfused placenta.

FIG. 3 is a cross-sectional schematic of a perfused placenta in a devicefor use as a bioreactor.

FIG. 4 is a selection scheme for sorting cells, including embryonic-likestem cells, retrieved from a perfused placenta.

5. DETAILED DESCRIPTION OF THE INVENTION

The applicant has unexpectedly discovered that the placenta after birthcontains quiescent cells which can be activated if the placenta isproperly processed after birth. For example, after expulsion from thewomb, the placenta is exsanguinated as quickly as possible to prevent orminimize apoptosis. Subsequently, as soon as possible afterexsanguination the placenta is perfused to remove blood, residual cells,proteins, factors and any other materials present in the organ.Materials debris may also be removed from the placenta. Perfusion isnormally continued with an appropriate perfusate for at least two tomore than twenty-four hours. In several additional embodiments theplacenta is perfused for at least 4, 6, 8, 10, 12, 14, 16, 18, 20, and22 hours. In other words, this invention is based at least in part onthe discovery that the cells of a post-partum placenta can be activatedby exsanguination and perfusion for a sufficient amount of time.Therefore, the placenta can readily be used as a rich and abundantsource of embryonic-like stem cells, which cells can be used forresearch, including drug discovery, treatment and prevention ofdiseases, in particular transplantation surgeries or therapies, and thegeneration of committed cells, tissues and organoids.

Further, surprisingly and unexpectedly the human placental stem cellsproduced by the exsanguinated, perfused and/or cultured placenta arepluripotent stem cells that can readily be differentiated into anydesired cell type.

The present invention relates to methods of treating and culturing anisolated placenta for use as a bioreactor for the production andpropagation of embryonic-like stem cells originating from the placentaor from exogenous sources. The present invention also relates to the useof a cultured placenta as a bioreactor to produce biological materials,including, but not limited to, antibodies, hormones, cytokines, growthfactors and viruses. The present invention also relates to methods ofcollecting and isolating the stem cells and biological materials fromthe cultured placenta.

The present invention relates to methods of perfusing and exsanguinatingan isolated placenta once it has been expunged from a uterus, to removeall residual cells. The invention further relates to culturing theisolated and exsanguinated placenta under the appropriate conditions toallow for the production and propagation of embryonic-like stem cells.

The present invention provides a method of extracting and recoveringembryonic-like stem cells, including, but not limited to pluripotent ormultipotent stem cells, from an exsanguinated human placenta.Embryonic-like stem cells have characteristics of embryonic stem cellsbut are not derived from the embryo. Such cells are as versatile (e.g.,pluripotent) as human embryonic stem cells. According to the methods ofthe invention, human placenta is used post-birth as the source ofembryonic-like stem cells.

According to the methods of the invention embryonic-like stem cells areextracted from a drained placenta by means of a perfusion technique thatutilizes either or both of the umbilical artery and umbilical vein. Theplacenta is preferably drained by exsanguination and collection ofresidual blood (e.g., residual umbilical cord blood). The drainedplacenta is then processed in such a manner as to establish an ex vivo,natural bioreactor environment in which resident embryonic-like stemcells within the parenchyma and extravascular space are recruited. Theembryonic-like stem cells migrate into the drained, emptymicrocirculation where, according to the methods of the invention, theyare collected, preferably by washing into a collecting vessel byperfusion.

5.1. Methods of Isolating and Culturing Placenta

5.1.1. Pretreatment of Placenta

According to the methods of the invention, a human placenta is recoveredshortly after its expulsion after birth and, in certain embodiments, thecord blood in the placenta is recovered. In certain embodiments, theplacenta is subjected to a conventional cord blood recovery process.Such cord blood recovery may be obtained commercially, e.g., LifeBankInc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and Cryocell. Thecord blood can be drained shortly after expulsion of the placenta.

Postpartum the placenta is drained of cord blood. The placenta storedmay be under sterile conditions and at either room temperature or at atemperature of 5 to 25° C. (centigrade). The placenta may be stored fora period of longer than forty eight hours, and preferably for a periodof four to twenty-four hours prior to perfusing the placenta to removeany residual cord blood.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of the cord bloodand/or drainage and perfusion. The placenta is preferably transported ina sterile, thermally insulated transport device (maintaining thetemperature of the placenta between 20-28° C.), for example, by placingthe placenta, with clamped proximal umbilical cord, in a sterilezip-lock plastic bag, which is then placed in an insulated container, asshown in FIGS. 2 a-e. Preferably, the placenta is delivered to thelaboratory four to twenty-four hours following delivery.

The placenta is preferably recovered after expulsion under asepticconditions, and stored in an anticoagulant solution at a temperature of5 to 25° C. (centigrade). Suitable anticoagulant solutions are wellknown in the art. For example, a solution of heparin or warfarin sodiumcan be used. In a preferred embodiment, the anticoagulant solutioncomprises a solution of heparin (1% w/w in 1:1000 solution). The drainedplacenta is preferably stored for no more than 36 hours before theembryonic-like stem cells are collected. The solution which is used toperfuse the placenta to remove residual cells can be the same solutionused to perfuse and culture the placenta for the recovery of stem cells.Any of these perfusates may be collected and used as a source ofembryonic-like stem cells.

In certain embodiments, the proximal umbilical cord is clamped,preferably within 4-5 cm (centimeter) of the insertion into theplacental disc prior to cord blood recovery. In other embodiments, theproximal umbilical cord is clamped after cord blood recovery but priorto further processing of the placenta.

Conventional techniques for the collection of cord blood may be used.Typically a needle or cannula is used, with the aid of gravity, to draincord blood from (i.e., exsanguinate) the placenta (Boyse et al., U.S.Pat. No. 5,192,553, issued Mar. 9, 1993; Boyse et al., U.S. Pat. No.5,004,681, issued Apr. 2, 1991; Anderson, U.S. Pat. No. 5,372,581,entitled Method and apparatus for placental blood collection, issuedDec. 13, 1994; Hessel et al., U.S. Pat. No. 5,415,665, entitledUmbilical cord clamping, cutting, and blood collecting device andmethod, issued May 16, 1995). The needle or cannula is usually placed inthe umbilical vein and the placenta is gently massaged to aid indraining cord blood from the placenta.

In a preferred embodiment, the placenta is recovered from a patient byinformed consent and a complete medical history of the patient prior to,during and after pregnancy is also taken and is associated with theplacenta. These medical records can be used to coordinate subsequent useof the placenta or the stem cells harvested therefrom. For example, thehuman placental stem cells can then easily be used for personalizedmedicine for the infant in question, the parents, siblings or otherrelatives. Indeed, the human placental stem cells are more versatilethan cord blood. However, it should be noted that the invention includesthe addition of human placental stem cells produced by theexsanguinated, perfused and/or cultured placenta to cord blood from thesame or different placenta and umbilical cord. The resulting cord bloodwill have an increased concentration/population of human stem cells andthereby is more useful for transplantation e.g. for bone marrowtransplantations.

5.1.2. Exsanguination of Placenta and Removal of Residual Cells

The invention provides a method for recovery of stem or progenitorcells, including, but not limited to embryonic-like stem cells, from aplacenta that is exsanguinated, i.e., completely drained of the cordblood remaining after birth and/or a conventional cord blood recoveryprocedure. According to the methods of the invention, the placenta isexsanguinated and perfused with a suitable aqueous perfusion fluid, suchas an aqueous isotonic fluid in which an anticoagulant (e.g., heparin,warfarin sodium) is dissolved. Such aqueous isotonic fluids forperfusion are well known in the art, and include, e.g., a 0.9 N sodiumchloride solution. The perfusion fluid preferably comprises theanticoagulant, such as heparin or warfarin sodium, at a concentrationthat is sufficient to prevent the formation of clots of any residualcord blood. In a specific embodiment, a concentration of from 1 to 100units of heparin is employed, preferably a concentration of 1 to 10units of heparin per ml is employed. In one embodiment, apoptosisinhibitors, such as free radical scavengers, in particular oxygen freeradical scavengers, can be used during and immediately afterexsanguination and then these agents can be washed from the placenta. Inaccordance with this embodiment of the invention, the isolated placentamay be stored under hypothermic conditions in order to prevent orinhibit apoptosis.

According to the methods of the invention, the placenta is exsanguinatedby passage of the perfusion fluid through either or both of theumbilical artery and umbilical vein, using a gravity flow into theplacenta. The placenta is preferably oriented (e.g., suspended) in sucha manner that the umbilical artery and umbilical vein are located at thehighest point of the placenta. In a preferred embodiment, the umbilicalartery and the umbilical vein are connected simultaneously, as shown inFIG. 1, to a pipette that is connected via a flexible connector to areservoir of the perfusion fluid. The perfusion fluid is passed into theumbilical vein and artery and collected in a suitable open vessel fromthe surface of the placenta that was attached to the uterus of themother during gestation. The perfusion fluid may also be introducedthrough the umbilical cord opening and allowed to flow or perculate outof openings in the wall of the placenta which interfaced with thematernal uterine wall.

In a preferred embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

In one embodiment, a sufficient amount of perfusion fluid is used thatwill result in removal of all residual cord blood and subsequentcollection or recovery of placental cells, including but not limited toembryonic-like stem cells and progenitor cells, that remain in theplacenta after removal of the cord blood.

It has been observed that when perfusion fluid is first collected from aplacenta during the exsanguination process, the fluid is colored withresidual red blood cells of the cord blood. The perfusion fluid tends tobecome clearer as perfusion proceeds and the residual cord blood cellsare washed out of the placenta. Generally from 30 to 100 ml (milliliter)of perfusion fluid is adequate to exsanguinate the placenta and torecover an initial population of embryonic-like cells from the placenta,but more or less perfusion fluid may be used depending on the observedresults.

5.1.3. Culturing Placenta

After exsanguination and a sufficient time of perfusion of the placenta,the embryonic-like stem cells are observed to migrate into theexsanguinated and perfused microcirculation of the placenta where,according to the methods of the invention, they are collected,preferably by washing into a collecting vessel by perfusion. Perfusingthe isolated placenta not only serves to remove residual cord blood butalso provide the placenta with the appropriate nutrients, includingoxygen. The placenta may be cultivated and perfused with a similarsolution which was used to remove the residual cord blood cells,preferably, without the addition of anticoagulant agents.

In certain embodiments of the invention, the drained, exsanguinatedplacenta is cultured as a bioreactor, i.e., an ex vivo system forpropagating cells or producing biological materials. The number ofpropagated cells or level of biological material produced in theplacental bioreactor is maintained in a continuous state of balancedgrowth by periodically or continuously removing a portion of a culturemedium or perfusion fluid that is introduced into the placentalbioreactor, and from which the propagated cells or the producedbiological materials may be recovered. Fresh medium or perfusion fluidis introduced at the same rate or in the same amount.

The number and type of cells propagated may easily be monitored bymeasuring changes in morphology and cell surface markers using standardcell detection techniques such as flow cytometry, cell sorting,immunocytochemistry (e.g., staining with tissue specific or cell-markerspecific antibodies) fluorescence activated cell sorting (FACS),magnetic activated cell sorting (MACS), by examination of the morphologyof cells using light or confocal microscopy, or by measuring changes ingene expression using techniques well known in the art, such as PCR andgene expression profiling.

In one embodiment, the cells may be sorted using a fluorescenceactivated cell sorter (FACS). Fluorescence activated cell sorting (FACS)is a well-known method for separating particles, including cells, basedon the fluorescent properties of the particles (Kamarch, 1987, MethodsEnzymol, 151:150-165). Laser excitation of fluorescent moieties in theindividual particles results in a small electrical charge allowingelectromagnetic separation of positive and negative particles from amixture. In one embodiment, cell surface marker-specific antibodies orligands are labeled with distinct fluorescent labels. Cells areprocessed through the cell sorter, allowing separation of cells based ontheir ability to bind to the antibodies used. FACS sorted particles maybe directly deposited into individual wells of 96-well or 384-wellplates to facilitate separation and cloning.

In another embodiment, magnetic beads can be used to separate cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody which specifically recognizes a cell-solidphase surface molecule or hapten. A magnetic field is then applied, tophysically manipulate the selected beads. The beads are then mixed withthe cells to allow binding. Cells are then passed through a magneticfield to separate out cells having cell surface markers. These cells canthen isolated and re-mixed with magnetic beads coupled to an antibodyagainst additional cell surface markers. The cells are again passedthrough a magnetic field, isolating cells that bound both theantibodies. Such cells can then be diluted into separate dishes, such asmicrotiter dishes for clonal isolation.

In preferred embodiments, the placenta to be used as a bioreactor isexsanguinated and washed under sterile conditions so that any adherentcoagulated and non-adherent cellular contaminants are removed. Theplacenta is then cultured or cultivated under aseptic conditions in acontainer or other suitable vessel, and perfused with perfusate solution(e.g., a normal saline solution such as phosphate buffered saline(“PBS”)) with or without an anticoagulant (e.g., heparin, warfarinsodium, coumarin, bishydroxycoumarin), and/or with or without anantimicrobial agent (e.g., β-mercaptoethanol (0.1 mM); antibiotics suchas streptomycin (e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml),amphotericin B (e.g., at 0.5 μg/ml).

The effluent perfusate comprises both circulated perfusate, which hasflowed through the placental circulation, and extravasated perfusate,which exudes from or passes through the walls of the blood vessels intothe surrounding tissues of the placenta. The effluent perfusate iscollected, and preferably, both the circulated and extravasatedperfusates are collected, preferably in a sterile receptacle.Alterations in the conditions in which the placenta is maintained andthe nature of the perfusate can be made to modulate the volume andcomposition of the effluent perfusate.

Cell types are then isolated from the collected perfusate by employingtechniques known by those skilled in the art, such as for example, butnot limited to density gradient centrifugation, magnet cell separation,flow cytometry, affinity cell separation or differential adhesiontechniques.

In one embodiment, a placenta is placed in a sterile basin and washedwith 500 ml of phosphate-buffered normal saline. The wash fluid is thendiscarded. The umbilical vein is then cannulated with a cannula, e.g., aTEFLON® or plastic cannula, that is connected to a sterile connectionapparatus, such as sterile tubing. The sterile connection apparatus isconnected to a perfusion manifold, as shown in FIG. 3. The containercontaining the placenta is then covered and the placenta is maintainedat room temperature (20-25° C.) for a desired period of time, preferablyfrom 2 to 24 hours, and preferably, no longer than 48 hours. Theplacenta may be perfused continually, with equal volumes of perfusateintroduced and effluent perfusate removed or collected. Alternatively,the placenta may be perfused periodically, e.g., at every 2 hours or at4, 8, 12, and 24 hours, with a volume of perfusate, e.g., 100 ml ofperfusate (sterile normal saline supplemented with or without 1000 u/lheparin and/or EDTA and/or CPDA (creatine phosphate dextrose)). In thecase of periodic perfusion, preferably equal volumes of perfusate areintroduced and removed from the culture environment of the placenta, sothat a stable volume of perfusate bathes the placenta at all times.

The effluent perfusate that escapes the placenta, e.g., at the oppositesurface of the placenta, is collected and processed to isolateembryonic-like stem cells, progenitor cells or other cells of interest.

Various media may be used as perfusion fluid for culturing orcultivating the placenta, such as DMEM, F-12, M199, RPMI, Fisher's,Iscore's, McCoy's and combinations thereof, supplemented with fetalbovine serum (FBS), whole human serum (WHS), or human umbilical cordserum collected at the time of delivery of the placenta. The sameperfusion fluid used to exsanguinate the placenta of residual cord bloodmay be used to culture or cultivate the placenta, without the additionof anticoagulant agents.

In certain embodiments, the embryonic-like stem cells are induced topropagate in the placenta bioreactor by introduction of nutrients,hormones, vitamins, growth factors, or any combination thereof, into theperfusion solution. Serum and other growth factors may be added to thepropagation perfusion solution or medium. Growth factors are usuallyproteins and include, but are not limited to: cytokines, lymphokines,interferons, colony stimulating factors (CSF's), interferons,chemokines, and interleukins. Other growth factors that may be usedinclude recombinant human hematopoietic growth factors includingligands, stem cell factors, thrombopoeitin (Tpo), granulocytecolony-stimulating factor (G-CSF), leukemia inhibitory factor, basicfibroblast growth factor, placenta derived growth factor and epidermalgrowth factor.

The growth factors introduced into the perfusion solution can stimulatethe propagation of undifferentiated embryonic-like stem cells, committedprogenitor cells, or differentiated cells (e.g., differentiatedhematopoietic cells). The growth factors can stimulate the production ofbiological materials and bioactive molecules including, but not limitedto, immunoglobulins, hormones, enzymes or growth factors as previouslydescribed.

In one embodiment of the invention, the placenta is used as a bioreactorfor propagating endogenous cells (i.e., cells that originate from theplacenta), including but not limited to, various kinds of pluripotentand/or totipotent embryonic-like stem cells and lymphocytes. To use theplacenta as a bioreactor, it may be cultured for varying periods of timeunder sterile conditions by perfusion with perfusate solution asdisclosed herein. In specific embodiments, the placenta is cultured forat least 12, 24, 36, or 48 hours, or for 3-5 days, 6-10 days, or for oneto two weeks. In a preferred embodiment, the placenta is cultured for 48hours. The cultured placenta should be “fed” periodically to remove thespent media, depopulate released cells, and add fresh media. Thecultured placenta should be stored under sterile conditions to reducethe possibility of contamination, and maintained under intermittent andperiodic pressurization to create conditions that maintain an adequatesupply of nutrients to the cells of the placenta. It should berecognized that the perfusing and culturing of the placenta can be bothautomated and computerized for efficiency and increased capacity.

In another embodiment, the placenta is processed to remove allendogenous proliferating cells, such as embryonic-like stem cells, andto allow foreign (i.e., exogenous) cells to be introduced and propagatedin the environment of the perfused placenta. The invention contemplatesa large variety of stem or progenitor cells that can be cultured in theplacental bioreactor, including, but not limited to, embryonic-like stemcells, mesenchymal stem cells, stromal cells, endothelial cells,hepatocytes, keratinocytes, and stem or progenitor cells for aparticular cell type, tissue or organ, including but not limited toneurons, myelin, muscle, blood, bone marrow, skin, heart, connectivetissue, lung, kidney, liver, and pancreas (e.g., pancreatic isletcells).

Sources of mesenchymal stem cells include bone marrow, embryonic yolksac, placenta, umbilical cord, fetal and adolescent skin, and blood.Bone marrow cells may be obtained from iliac crest, femora, tibiae,spine, rib or other medullary spaces.

Methods for the selective destruction, ablation or removal ofproliferating or rapidly dividing cells from a tissue or organ arewell-known in the art, e.g., methods of cancer or tumor treatment. Forexample, the perfused placenta may be irradiated with electromagnetic,UV, X-ray, gamma- or beta-radiation to eradicate all remaining viable,endogenous cells. The foreign cells to be propagated are introduced intothe irradiated placental bioreactor, for example, by perfusion.

5.2. Collection of Cells from the Placenta

As disclosed above, after exsanguination and perfusion of the placenta,embryonic-like stem cells migrate into the drained, emptymicrocirculation where, according to the methods of the invention, theyare collected, preferably by collecting the effluent perfusate in acollecting vessel.

In preferred embodiments, cells cultured in the placenta are isolatedfrom the effluent perfusate using techniques known by those skilled inthe art, such as, for example, density gradient centrifugation, magnetcell separation, flow cytometry, or other cell separation or sortingmethods well known in the art, and sorted, for example, according to thescheme shown in FIG. 4.

In a specific embodiment, cells collected from the placenta arerecovered from the effluent perfusate by centrifugation at 5000×g for 15minutes at room temperature, which separates cells from contaminatingdebris and platelets. The cell pellets are resuspended in IMDMserum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL,NY). The total mononuclear cell fraction was isolated using Lymphoprep(Nycomed Pharma, Oslo, Norway) according to the manufacturer'srecommended procedure and the mononuclear cell fraction was resuspended.Cells were counted using a hemocytometer. Viability was evaluated bytrypan blue exclusion. Isolation of cells is achieved by “differentialtrypsinization,” using a solution of 0.05% trypsin with 0.2% EDTA(Sigma, St. Louis Mo.). Differential trypsinization was possible becausefibroblastoid cells detached from plastic surfaces within about fiveminutes whereas the other adherent populations required more than 20-30minutes incubation. The detached fibroblastoid cells were harvestedfollowing trypsinization and trypsin neutralization, using TrypsinNeutralizing Solution (TNS, BioWhittaker). The cells were washed inH.DMEM and resuspended in MSCGM.

In another embodiment, the isolated placenta is perfused for a period oftime without collecting the perfusate, such that the placenta may beperfused for 2, 4, 6, 8, 10, 12, 20 and 24 hours or even days before theperfusate is collected.

In another embodiment, cells cultured in the placenta bioreactor areisolated from the placenta by physically dissecting the cells away fromthe placenta.

In another embodiment, cells cultured in the placenta bioreactor areisolated from the placenta by dissociating the tissues of the placentaor a portion thereof, and recovering the cultured cells by art-knowncell separation or sorting methods such as density gradientcentrifugation, magnet cell separation, flow cytometry, etc.

In a preferred embodiment, perfusion of the placenta and collection ofeffluent perfusate is repeated once or twice during the culturing of theplacenta, until the number of recovered nucleated cells falls below 100cells/ml. The perfusates are pooled and subjected to lightcentrifugation to remove platelets, debris and de-nucleated cellmembranes. The nucleated cells are then isolated by Ficoll-Hypaquedensity gradient centrifugation and after washing, resuspended inH.DMEM. For isolation of the adherent cells, aliquots of 5-10×10⁶ cellsare placed in each of several T-75 flasks and cultured with commerciallyavailable Mesenchymal Stem Cell Growth Medium (MSCGM) obtained fromBioWhittaker, and placed in a tissue culture incubator (37° C., 5% CO₂).After 10 to 15 days, non-adherent cells are removed from the flasks bywashing with PBS. The PBS is then replaced by MSCGM. Flasks arepreferably examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

In other embodiments, the cells collected from the placenta arecryopreserved for use at a later time. Methods for cryopreservation ofcells, such as stem cells, are well known in the art, for example,cryopreservation using the methods of Boyse et al. U.S. Pat. No.5,192,553, issued Mar. 9, 1993) or Hu et al. (WO 00/73421, publishedDec. 7, 2000).

5.3. Cell Populations Obtained from or Cultured in Placenta

Embryonic-like stem cells obtained in accordance with the methods of theinvention may include pluripotent cells, i.e., cells that have completedifferentiation versatility, that are self-renewing, and can remaindormant or quiescent within tissue. The stem cells which may be obtainedfrom the placenta include embryonic-like stem cells, multipotent cells,committed progenitor cells, and fibroblastoid cells.

The first collection of blood from the placenta is referred to as cordblood which contains predominantly CD34+ and CD38+ hematopoieticprogenitor cells. Within the first twenty-four hours of post-partumperfusion, high concentrations of CD34+ and CD38− hematopoieticprogenitor cells may be isolated from the placenta, along with highconcentrations of CD34− and CD38+ hematopoietic progenitor cells. Afterabout twenty-four hours of perfusion, high concentrations of CD34− andCD38− cells can be isolated from the placenta along with theaforementioned cells. The isolated perfused placenta of the inventionprovides a source of large quantities of stem cells enriched for CD34+and CD38− stem cells and CD34− and CD38+ stem cells. The isolatedplacenta which has been perfused for twenty-four hours or more providesa source of large quantities of stem cells enriched for CD34− and CD38−stem cells.

In a preferred embodiment, embryonic-like stem cells obtained by themethods of the invention are viable, quiescent, pluripotent stem cellsthat exist within a full-term human placenta and that can be recoveredfollowing successful birth and placental expulsion, resulting in therecovery of as many as one billion nucleated cells, which yield 50-100million multipotent and pluripotent stem cells.

The human placental stem cells provided by the placenta are surprisinglyembryonic-like, for example, the presence of the following cell surfacemarkers have been identified for these cells: SSEA3−, SSEA4−, OCT-4+ andABC-p⁺. Preferably, the embryonic-like stem cells of the invention arecharacterized by the presence of OCT-4+ and ABC-p+ cell surface markers.Thus, the invention encompasses stem cells which have not been isolatedor otherwise obtained from an embryonic source but which can beidentified by the following markers: SSAE3−, SSAE4−, OCT-4+ and ABC-p+.In one embodiment, the human placental stem cells do not express MHCClass 2 antigens.

The stem cells isolated from the placenta are homogenous, and sterile.Further, the stem cells are readily obtained in a form suitable foradministration to humans, i.e., they are of pharmaceutical grade.

Preferred embryonic-like stem cells obtained by the methods of theinvention may be identified by the presence of the following cellsurface markers: OCT-4+ and ABC-pt. Further, the invention encompassesembryonic stem cells having the following markers: CD10+, CD38−, CD29+,CD34−, CD44+, CD45−, CD54+, CD90+, SH2+, SH3+, SH4+, SSEA3−, SSEA4−,OCT-4+, and ABC-p+. Such cell surface markers are routinely determinedaccording to methods well known in the art, e.g. by flow cytometry,followed by washing and staining with an anti-cell surface markerantibody. For example, to determine the presence of CD-34 or CD-38,cells may be washed in PBS and then double-stained with anti-CD34phycoerythrin and anti-CD38 fluorescein isothiocyanate (BectonDickinson, Mountain View, Calif.).

In another embodiment, cells cultured in the placenta bioreactor areidentified and characterized by a colony forming unit assay, which iscommonly known in the art, such as Mesen Cult™ medium (stem cellTechnologies, Inc., Vancouver British Columbia)

The embryonic-like stem cells obtained by the methods of the inventionmay be induced to differentiate along specific cell lineages, includingadipogenic, chondrogenic, osteogenic, hematopoietic, myogenic,vasogenic, neurogenic, and hepatogenic. In certain embodiments,embryonic-like stem cells obtained according to the methods of theinvention are induced to differentiate for use in transplantation and exvivo treatment protocols. In certain embodiments, embryonic-like stemcells obtained by the methods of the invention are induced todifferentiate into a particular cell type and genetically engineered toprovide a therapeutic gene product. In a specific embodiment,embryonic-like stem cells obtained by the methods of the invention areincubated with a compound in vitro that induces it to differentiate,followed by direct transplantation of the differentiated cells to asubject. Thus, the invention encompasses methods of differentiating thehuman placental stem cells using standard culturing media. Further, theinvention encompasses hematopoietic cells, neuron cells, fibroblastcells, strand cells, mesenchymal cells and hepatic cells.

Embryonic-like stem cells may also be further cultured after collectionfrom the placenta using methods well known in the art, for example, byculturing on feeder cells, such as irradiated fibroblasts, obtained fromthe same placenta as the embryonic-like stem cells or from other humanor nonhuman sources, or in conditioned media obtained from cultures ofsuch feeder cells, in order to obtain continued long-term cultures ofembryonic-like stem cells. The embryonic-like stem cells may also beexpanded, either within the placenta before collection from theplacental bioreactor or in vitro after recovery from the placenta. Incertain embodiments, the embryonic-like stem cells to be expanded areexposed to, or cultured in the presence of, an agent that suppressescellular differentiation. Such agents are well-known in the art andinclude, but are not limited to, human Delta-1 and human Serrate-1polypeptides (see, Sakano et al., U.S. Pat. No. 6,337,387 entitled“Differentiation-suppressive polypeptide”, issued Jan. 8, 2002),leukemia inhibitory factor (LIF) and stem cell factor. Methods for theexpansion of cell populations are also known in the art (see e.g.,Emerson et al., U.S. Pat. No. 6,326,198 entitled “Methods andcompositions for the ex vivo replication of stem cells, for theoptimization of hematopoietic progenitor cell cultures, and forincreasing the metabolism, GM-CSF secretion and/or IL-6 secretion ofhuman stromal cells”, issued Dec. 4, 2001; Kraus et al., U.S. Pat. No.6,338,942, entitled “Selective expansion of target cell populations”,issued Jan. 15, 2002).

The embryonic-like stem cells may be assessed for viability,proliferation potential, and longevity using standard techniques knownin the art, such as trypan blue exclusion assay, fluorescein diacetateuptake assay, propidium iodide uptake assay (to assess viability); andthymidine uptake assay, MTT cell proliferation assay (to assessproliferation). Longevity may be determined by methods well known in theart, such as by determining the maximum number of population doubling inan extended culture.

In certain embodiments, the differentiation of stem cells or progenitorcells that are cultivated in the exsanguinated, perfused and/or culturedplacenta is modulated using an agent or pharmaceutical compositionscomprising a dose and/or doses effective upon single or multipleadministration, to exert an effect sufficient to inhibit, modulateand/or regulate the differentiation of a cell collected from theplacenta.

Agents that can induce stem or progenitor cell differentiation are wellknown in the art and include, but are not limited to, Ca²⁺, EGF, aFGF,bFGF, PDGF, keratinocyte growth factor (KGF), TGF-β, cytokines (e.g.,IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin, hormones (e.g.,androgen, estrogen, insulin, prolactin, triiodothyronine,hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF,matrix elements (e.g., collagen, laminin, heparan sulfate, Matrigel™),or combinations thereof.

Agents that suppress cellular differentiation are also well-known in theart and include, but are not limited to, human Delta-1 and humanSerrate-1 polypeptides (see, Sakano et al., U.S. Pat. No. 6,337,387entitled “Differentiation-suppressive polypeptide”, issued Jan. 8,2002), leukemia inhibitory factor (LIF), and stem cell factor.

The agent used to modulate differentiation can be introduced into theplacental bioreactor to induce differentiation of the cells beingcultured in the placenta. Alternatively, the agent can be used tomodulate differentiation in vitro after the cells have been collected orremoved from the placenta.

Determination that a stem cell has differentiated into a particular celltype may be accomplished by methods well-known in the art, e.g.,measuring changes in morphology and cell surface markers usingtechniques such as flow cytometry or immunocytochemistry (e.g., stainingcells with tissue-specific or cell-marker specific antibodies), byexamination of the morphology of cells using light or confocalmicroscopy, or by measuring changes in gene expression using techniqueswell known in the art, such as PCR and gene-expression profiling.

In another embodiment, the cells cultured in the placenta are stimulatedto produce bioactive molecules, such as immunoglobulins, hormones,enzymes.

In another embodiment, the cells cultured in the placenta are stimulatedto proliferate, for example, by administration of erythropoietin,cytokines, lymphokines, interferons, colony stimulating factors (CSF's),interferons, chemokines, interleukins, recombinant human hematopoieticgrowth factors including ligands, stem cell factors, thrombopoeitin(Tpo), interleukins, and granulocyte colony-stimulating factor (G-CSF)or other growth factors.

In another embodiment, cells cultured in the placenta are geneticallyengineered either prior to, or after collection from, the placenta,using, for example, a viral vector such as an adenoviral or retroviralvector, or by using mechanical means such as liposomal or chemicalmediated uptake of the DNA.

A vector containing a transgene can be introduced into a cell ofinterest by methods well known in the art, e.g., transfection,transformation, transduction, electroporation, infection,microinjection, cell fusion, DEAE dextran, calcium phosphateprecipitation, liposomes, LIPOFECTIN™, lysosome fusion, syntheticcationic lipids, use of a gene gun or a DNA vector transporter, suchthat the transgene is transmitted to daughter cells, e.g., the daughterembryonic-like stem cells or progenitor cells produced by the divisionof an embryonic-like stem cell. For various techniques fortransformation or transfection of mammalian cells, see Keown et al.,1990, Methods Enzymol. 185: 527-37; Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, N.Y.

Preferably, the transgene is introduced using any technique, so long asit is not destructive to the cell's nuclear membrane or other existingcellular or genetic structures. In certain embodiments, the transgene isinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is commonly known andpracticed in the art.

For stable transfection of cultured mammalian cells, such as cellsculture in a placenta, only a small fraction of cells may integrate theforeign DNA into their genome. The efficiency of integration dependsupon the vector and transfection technique used. In order to identifyand select integrants, a gene that encodes a selectable marker (e.g.,for resistance to antibiotics) is generally introduced into the hostembryonic-like stem cell along with the gene sequence of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die). Such methods are particularlyuseful in methods involving homologous recombination in mammalian cells(e.g., in embryonic-like stem cells) prior to introduction ortransplantation of the recombinant cells into a subject or patient.

A number of selection systems may be used to select transformed hostembryonic-like cells. In particular, the vector may contain certaindetectable or selectable markers. Other methods of selection include butare not limited to selecting for another marker such as: the herpessimplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski,1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78: 2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147).

The transgene may integrate into the genome of the cell of interest,preferably by random integration. In other embodiments the transgene mayintegrate by a directed method, e.g., by directed homologousrecombination (i.e., “knock-in” or “knock-out” of a gene of interest inthe genome of cell of interest), Chappel, U.S. Pat. No. 5,272,071; andPCT publication No. WO 91/06667, published May 16, 1991; U.S. Pat. No.5,464,764; Capecchi et al., issued Nov. 7, 1995; U.S. Pat. No.5,627,059, Capecchi et al. issued, May 6, 1997; U.S. Pat. No. 5,487,992,Capecchi et al., issued Jan. 30, 1996).

Methods for generating cells having targeted gene modifications throughhomologous recombination are known in the art. The construct willcomprise at least a portion of a gene of interest with a desired geneticmodification, and will include regions of homology to the target locus,i.e., the endogenous copy of the targeted gene in the host's genome. DNAconstructs for random integration, in contrast to those used forhomologous recombination, need not include regions of homology tomediate recombination. Markers can be included in the targetingconstruct or random construct for performing positive and negativeselection for insertion of the transgene.

To create a homologous recombinant cell, e.g., a homologous recombinantembryonic-like stem cell, endogenous placental cell or exogenous cellcultured in the placenta, a homologous recombination vector is preparedin which a gene of interest is flanked at its 5′ and 3′ ends by genesequences that are endogenous to the genome of the targeted cell, toallow for homologous recombination to occur between the gene of interestcarried by the vector and the endogenous gene in the genome of thetargeted cell. The additional flanking nucleic acid sequences are ofsufficient length for successful homologous recombination with theendogenous gene in the genome of the targeted cell. Typically, severalkilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector. Methods for constructing homologous recombination vectorsand homologous recombinant animals from recombinant stem cells arecommonly known in the art (see, e.g., Thomas and Capecchi, 1987, Cell51: 503; Bradley, 1991, Curr. Opin. Bio/Technol. 2: 823-29; and PCTPublication Nos. WO 90/11354, WO 91/01140, and WO 93/04169.

In one embodiment, the genome of an exogenous cell cultured in theplacenta according to the methods of the invention is a target of genetargeting via homologous recombination or via random integration.

In a specific embodiment, the methods of Bonadio et al. (U.S. Pat. No.5,942,496, entitled Methods and compositions for multiple gene transferinto bone cells, issued Aug. 24, 1999; and PCT WO95/22611, entitledMethods and compositions for stimulating bone cells, published Aug. 24,1995) are used to introduce nucleic acids into a cell of interest, suchas a stem cell, progenitor cell or exogenous cell cultured in theplacenta, e.g., bone progenitor cells.

5.4 Uses of Cultured Placenta as a Bioreactor

Exsanguinated and/or cultured placental cells can be used as abioreactor for the cultivation of cells, tissues, and organs. Theplacental mesoderm provides an ideal stromal environment, including anabundance of small molecules and growth factors, lipopolysaccharides,and extracellular matrix proteins, necessary for organogenesis andtissue neogenesis.

In one embodiment, the invention provides a method of utilizing theisolated perfused placenta as a bioreactor for the propagation ofexogenous cells. In accordance with this embodiment, the inventionrelates to an isolated placenta which contains a cell not derived fromthe placenta, wherein the engraftment of said cell into the placenta maystimulate the placenta to produce embryonic-like stem cells, or whereinthe engrafted cell produces signals, such as cytokines and growthfactors, which may stimulate the placenta to produce stem cells. Theplacenta may be engrafted with cells not placental in origin obtainedfrom the parents, siblings or other blood relatives of the infantassociated with the placenta. In another embodiment, the isolatedplacenta may be engrafted with cells not placental in origin obtainedfrom an individual whom is not the infant, nor related to the infant.Likewise, the cells, tissues, organoids and organs, which are propagatedand cultivated in the placenta may be transplanted into the infantassociated with the placenta, the parents, siblings or other bloodrelatives of said infant or into an individual not related to theinfant.

In one embodiment of the invention, the placenta can be populated withany particular cell type and used as a bioreactor for ex vivocultivation of cells, tissues or organs. Such cells, tissue or organcultures may be harvested used in transplantation and ex vivo treatmentprotocols. In this embodiment, the placenta is processed to remove allendogenous cells and to allow foreign (i.e., exogenous) cells to beintroduced and propagated in the environment of the perfused placenta.Methods for removal of the endogenous cells are well-known in the art.For example, the perfused placenta is irradiated with electromagnetic,V, X-ray, gamma- or beta-radiation to eradicate all remaining viable,endogenous cells. In one embodiment, sub-lethal exposure to radiationse.g., 500 to 1500 CGγ can be used to preserve the placenta but eradicateundesired cells. For international on lethal v. non-lethal ionizingradiation (see Chapter 5 “Biophysical and Biological Effects of IonizingRadiation” from the United States Department of Defense The foreigncells of interest to be propagated in the irradiated placentalbioreactor are then introduced, for example, by vascular perfusion ordirect intra-parenchymal injection.

In another embodiment, the bioreactor may be used to produce andpropagate novel chimeric cells, tissues, or organs. Such chimeras may becreated using placental cells and one or more additional cell types asstarting materials in a bioreactor. The interaction, or “cross-talk”between the different cell types can induce expression patterns distinctfrom either of the starting cell types. In one embodiment, for example,an autologous chimera is generated by propagating a patient's autologousplacental cells in a bioreactor with another cell type derived from thesame patient. In another embodiment, for example, a heterologous chimeramay be generated by addition of a patient's cells, i.e., blood cells, toa bioreactor having heterologous placental cells. In yet anotherembodiment, the placental cells may be derived from a patient, and asecond cell type from a second patient. Chimeric cells are thenrecovered having a different phenotypic and/or genetic characteristicsfrom either of the starting cells. In a specific embodiment, theheterologous cells are of the same haplotype, and the chimeric cells arereintroduced into the patient.

In other embodiments, the bioreactor may be used for enhanced growth ofa particular cell type, whether native or synthetic in origin, or forthe production of a cell-type specific product. For example, in oneembodiment, the placental bioreactor may be used to stimulate pancreaticislet cells to produce insulin. The bioreactor is particularlyadvantageous for production of therapeutic mammalian proteins, whosetherapeutic efficacy can be dependent upon proper post-translationalmodification. Thus, the bioreactor is useful for the production oftherapeutic proteins, growth factors, cytokines, and other natural orrecombinant therapeutic molecules, such as but not limited to,erythropoietin, interleukins, and interferons.

In another embodiment, the bioreactor may be used to propagategenetically engineered cells to provide a therapeutic gene product, andemployed for large-scale production of the recombinant product. In oneembodiment, for example, the reactor may be used to enhance antibodyproduction. The placenta may be populated with antibody-producing cells,such as hybridomas, which produce a specific monoclonal antibodies,which are homogeneous populations of antibodies to a particular antigen.Hybridomas may be obtained by any technique, including, but not limitedto, the hybridoma technique of Kohler and Milstein (1975, Nature 256,495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4, 72; Cole et al.,1983, Proc. Natl. Acad. Sci. USA 80, 2026-2030), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). The mAb-producing hybridomas may becultivated in the bioreactor to produce high titers of mAbs.

Alternatively, where an antigen is unknown, the bioreactor may be usedto generate antibodies specific for a particular cell-type, which maythen be used identify the unknown antigen. For example, antibodies maybe generated against an unknown tumor-specific antigen in a cancerpatient by culturing a whole blood specimen from a cancer patient,expanding the cells in a bioreactor, and then screened for antibodiesthat specifically react against a patient's tumor cells.

In another embodiment, the bioreactor may be used to produce viruses inculture and for screening for antiviral agents in culture. This methodis of particular interest for those viruses, such as parvovirus andhuman immunodeficiency virus, which are difficult to propagate in cellculture conditions.

The bioreactor may also be used as a support for screening fortherapeutic molecules which modulate the activity of a particular celltype, such as the activity or expression of a gene product of interestor the activation of a signal transduction pathway. In this embodiment,a cell type of interest may be cultured and expanded in the bioreactor.The cell may be naturally occurring cell, or a cell engineered toexpress a recombinant gene product. The bioreactor is then be contactedwith candidate therapeutic molecules, such as small molecules,nonpeptides, antibodies, etc., or libraries of such candidatetherapeutic molecules. The cells are then analyzed for a change in adesired activity in the presence or absence of the candidate therapeuticmolecule. For example, such desired activity might be an increase ordecrease in growth rate, and change in gene expression, or a change inbinding or uptake of the candidate therapeutic molecule.

Several types of methods are likely to be particularly convenient and/oruseful for screening test agents. These include, but are not limited to,methods which measure binding of a compound, methods which measure achange in the ability cells to interact with an antibody or ligand, andmethods which measure the activity or expression of “reporter” protein,that is, an enzyme or other detectable or selectable protein, which hasbeen placed under the control of a control region of interest. Thus, ina preferred embodiment, both naturally occurring and/or syntheticcompounds (e.g., libraries of small molecules or peptides), may bescreened for therapeutic activity. The screening assays can be used toidentify compounds and compositions including peptides and organic,non-protein molecules that modulate a cell-type specific activity.Recombinant, synthetic, and otherwise exogenous compounds may havebinding capacity and, therefore, may be candidates for pharmaceuticalagents. Alternatively, the proteins and compounds include endogenouscellular components which interact with the identified genes andproteins in vivo. Such endogenous components may provide new targets forpharmaceutical and therapeutic interventions.

In another embodiment of the invention, the placenta is used as abioreactor for propagating endogenous cells (i.e., cells that originatefrom the placenta), including but not limited to, various kinds ofpluripotent and/or totipotent embryonic-like stem cells and lymphocytes.In one embodiment, the placenta is incubated for varying periods of timewith perfusate solution as disclosed herein. Such endogenous cells ofplacental origin may be transformed to recombinantly express a gene ofinterest, to express mutations, and/or may be engineered to delete agenetic locus, using “knock out” technology. For example, an endogenoustarget gene may be deleted by inactivating or “knocking out” the targetgene or its promoter using targeted homologous recombination (e.g., seeSmithies, et al., 1985, Nature 317, 230-234; Thomas & Capecchi, 1987,Cell 51, 503-512; Thompson, et al., 1989, Cell 5, 313-321; each of whichis incorporated by reference herein in its entirety). For example, amutant, non-functional target gene (or a completely unrelated DNAsequence) flanked by DNA homologous to the endogenous target gene(either the coding regions or regulatory regions of the target gene) canbe used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express the target gene invivo. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the target gene. Suchapproaches may be used to remove, replace, or alter gene expression ofinterest in cells, tissue, and/or organs. This approach may be used toalter the phenotype of a cell, tissue, or organ, which may then beintroduced into a human subject.

In other embodiments, a placenta cell may be induced to differentiateinto a particular cell type, either ex vivo or in vivo. For example,pluripotent embryonic-like stem cells may be injected into a damagedorgan, and for organ neogenesis and repair of injury in vivo. Suchinjury may be due to such conditions and disorders including, but notlimited to, myocardial infarction, seizure disorder, multiple sclerosis,stroke, hypotension, cardiac arrest, ischemia, inflammation, age-relatedloss of cognitive function, radiation damage, cerebral palsy,neurodegenerative disease, Alzheimer's disease, Parkinson's disease,Leigh disease, AIDS dementia, memory loss, amyotrophic lateralsclerosis, ischemic renal disease, brain or spinal cord trauma,heart-lung bypass, glaucoma, retinal ischemia, or retinal trauma.

The embryonic-like stem cells isolated from the placenta may be used, inspecific embodiments, in autologous or heterologous enzyme replacementtherapy to treat specific diseases or conditions, including, but notlimited to lysosomal storage diseases, such as Tay-Sachs, Niemann-Pick,Fabry's, Gaucher's, Hunter's, and Hurler's syndromes, as well as othergangliosidoses, mucopolysaccharidoses, and glycogenoses.

In other embodiments, the cells may be used as autologous orheterologous transgene carriers in gene therapy to correct inborn errorsof metabolism, adrenoleukodystrophy, cystic fibrosis, glycogen storagedisease, hypothyroidism, sickle cell anemia, Pearson syndrome, Pompe'sdisease, phenylketonuria (PKU), porphyrias, maple syrup urine disease,homocystinuria, mucopolysaccharide nosis, chronic granulomatous diseaseand tyrosinemia and Tay-Sachs disease or to treat cancer, tumors orother pathological conditions.

In other embodiments, the cells may be used in autologous orheterologous tissue regeneration or replacement therapies or protocols,including, but not limited to treatment of corneal epithelial defects,cartilage repair, facial dermabrasion, mucosal membranes, tympanicmembranes, intestinal linings, neurological structures (e.g., retina,auditory neurons in basilar membrane, olfactory neurons in olfactoryepithelium), burn and wound repair for traumatic injuries of the skin,or for reconstruction of other damaged or diseased organs or tissues.

The large numbers of embryonic-like stem cells and/or progenitorobtained using the methods of the invention would, in certainembodiments, reduce the need for large bone marrow donations.Approximately 1×10⁸ to 2×10⁸ bone marrow mononuclear cells per kilogramof patient weight must be infused for engraftment in a bone marrowtransplantation (i.e., about 70 ml of marrow for a 70 kg donor). Toobtain 70 ml requires an intensive donation and significant loss ofblood in the donation process. In a specific embodiment, cells from asmall bone marrow donation (e.g., 7-10 ml) could be expanded bypropagation in a placental bioreactor before infusion into a recipient.

Furthermore, a small number of stem cells and progenitor cells normallycirculate in the blood stream. In another embodiment, such exogenousstem cells or exogenous progenitor cells are collected by pheresis, aprocedure in which blood is withdrawn, one or more components areselectively removed, and the remainder of the blood is reinfused intothe donor. The exogenous cells recovered by pheresis are expanded bypropagation in a placental bioreactor, thus eliminating the need forbone marrow donation entirely.

In another embodiment, expansion of exogenous cells in a placentalbioreactor is used as a supplemental treatment in addition tochemotherapy. Most chemotherapy agents used to target and destroy cancercells act by killing all proliferating cells, i.e., cells going throughcell division. Since bone marrow is one of the most activelyproliferating tissues in the body, hematopoietic stem cells arefrequently damaged or destroyed by chemotherapy agents and inconsequence, blood cell production is diminishes or ceases. Chemotherapymust be terminated at intervals to allow the patient's hematopoieticsystem to replenish the blood cell supply before resuming chemotherapy.It may take a month or more for the formerly quiescent stem cells toproliferate and increase the white blood cell count to acceptable levelsso that chemotherapy may resume (when again, the bone marrow stem cellsare destroyed).

While the blood cells regenerate between chemotherapy treatments,however, the cancer has time to grow and possibly become more resistantto the chemotherapy drugs due to natural selection. Therefore, thelonger chemotherapy is given and the shorter the duration betweentreatments, the greater the odds of successfully killing the cancer. Toshorten the time between chemotherapy treatments, embryonic-like stemcells or progenitor cells collected according to the methods of theinvention could be introduced into the patient. Such treatment wouldreduce the time the patient would exhibit a low blood cell count, andwould therefore permit earlier resumption of the chemotherapy treatment.

The embryonic-like stem cells, progenitor cells, foreign cells, orengineered cells obtained from a placenta according to the methods ofthe invention can be used in the manufacture of a tissue or organ invivo. The methods of the invention encompass using cells obtained fromthe placenta, e.g., embryonic-like stem cells, progenitor cells, orforeign stem or progenitor cells, to seed a matrix and to be culturedunder the appropriate conditions to allow the cells to differentiate andpopulate the matrix. The tissues and organs obtained by the methods ofthe invention may be used for a variety of purposes, including researchand therapeutic purposes.

5.5 Uses of Embryonic-like Stem Cells

The embryonic-like stem cells of the invention can be used for a widevariety of therapeutic protocols in which a tissue or organ of the bodyis augmented, repaired or replaced by the engraftment, transplantationor infusion of a desired cell population, such as a stem cell orprogenitor cell population. The embryonic-like stem cells of theinvention can be used to replace or augment existing tissues, tointroduce new or altered tissues, or to join together biological tissuesor structures. The embryonic-like stem cells of the invention can alsobe substituted for embryonic stem cells in therapeutic protocols inwhich embryonic stem cells would be typically be used.

In a preferred embodiment of the invention, embryonic-like stem cellsand other stem cells from the placenta may be used as autologous andallogenic, including matched and mismatched HLA type hematopoietictransplants. In accordance with the use of embryonic-like stem cells asallogenic hematopoietic transplants it may be necessary to treat thehost to reduce immunological rejection of the donor cells, such as thosedescribed in U.S. Pat. No. 5,800,539, issued Sep. 1, 1998; and U.S. Pat.No. 5,806,529, issued Sep. 15, 1998, both of which are incorporatedherein by reference.

For example, embryonic-like stem cells of the invention can be used intherapeutic transplantation protocols, e.g., to augment or replace stemor progenitor cells of the liver, pancreas, kidney, lung, nervoussystem, muscular system, bone, bone marrow, thymus, spleen, mucosaltissue, gonads, or hair.

Embryonic-like stem cells may be used instead of specific classes ofprogenitor cells (e.g., chondrocytes, hepatocytes, hematopoietic cells,pancreatic parenchymal cells, neuroblasts, muscle progenitor cells,etc.) in therapeutic or research protocols in which progenitor cellswould typically be used.

Embryonic-like stem cells of the invention can be used for augmentation,repair or replacement of cartilage, tendon, or ligaments. For example,in certain embodiments, prostheses (e.g., hip prostheses) are coatedwith replacement cartilage tissue constructs grown from embryonic-likestem cells of the invention. In other embodiments, joints (e.g., knee)are reconstructed with cartilage tissue constructs grown fromembryonic-like stem cells. Cartilage tissue constructs can also beemployed in major reconstructive surgery for different types of joints(for protocols, see e.g., Resnick, D., and Niwayama, G., eds., 1988,Diagnosis of Bone and Joint Disorders, 2d ed., W. B. Saunders Co.).

The embryonic-like stem cells of the invention can be used to repairdamage of tissues and organs resulting from disease. In such anembodiment, a patient can be administered embryonic-like stem cells toregenerate or restore tissues or organs which have been damaged as aconsequence of disease, e.g., enhance immune system followingchemotherapy or radiation, repair heart tissue following myocardialinfarction.

The embryonic-like stem cells of the invention can be used to augment orreplace bone marrow cells in bone marrow transplantation. Humanautologous and allogenic bone marrow transplantation are currently usedas therapies for diseases such as leukemia, lymphoma and otherlife-threatening disorders. The drawback of these procedures, however,is that a large amount of donor bone marrow must be removed to insurethat there is enough cells for engraftment.

The embryonic-like stem cells collected according to the methods of theinvention can provide stem cells and progenitor cells that would reducethe need for large bone marrow donation. It would also be, according tothe methods of the invention, to obtain a small marrow donation and thenexpand the number of stem cells and progenitor cells culturing andexpanding in the placenta before infusion or transplantation into arecipient.

The embryonic-like stem cells isolated from the placenta may be used, inspecific embodiments, in autologous or heterologous enzyme replacementtherapy to treat specific diseases or conditions, including, but notlimited to lysosomal storage diseases, such as Tay-Sachs, Niemann-Pick,Fabry's, Gaucher's, Hunter's, Hurler's syndromes, as well as othergangliosidoses, mucopolysaccharidoses, and glycogenoses.

In other embodiments, the cells may be used as autologous orheterologous transgene carriers in gene therapy to correct inborn errorsof metabolism such as adrenoleukodystrophy, cystic fibrosis, glycogenstorage disease, hypothyroidism, sickle cell anemia, Pearson syndrome,Pompe's disease, phenylketonuria (PKU), and Tay-Sachs disease,porphyrias, maple syrup urine disease, homocystinuria,mucopolypsaccharide nosis, chronic granulomatous disease, andtyrosinemia. or to treat cancer, tumors or other pathologicalconditions.

In other embodiments, the cells may be used in autologous orheterologous tissue regeneration or replacement therapies or protocols,including, but not limited to treatment of corneal epithelial defects,cartilage repair, facial dermabrasion, mucosal membranes, tympanicmembranes, intestinal linings, neurological structures (e.g., retina,auditory neurons in basilar membrane, olfactory neurons in olfactoryepithelium), burn and wound repair for traumatic injuries of the skin,scalp (hair) transplantation, or for reconstruction of other damaged ordiseased organs or tissues.

The large numbers of embryonic-like stem cells and/or progenitorobtained using the methods of the invention would, in certainembodiments, reduce the need for large bone marrow donations.Approximately 1×10⁸ to 2×10⁸ bone marrow mononuclear cells per kilogramof patient weight must be infused for engraftment in a bone marrowtransplantation (i.e., about 70 ml of marrow for a 70 kg donor). Toobtain 70 ml requires an intensive donation and significant loss ofblood in the donation process. In a specific embodiment, cells from asmall bone marrow donation (e.g., 7-10 ml) could be expanded bypropagation in a placental bioreactor before infusion into a recipient.

In another embodiment, the embryonic-like stem cells can be used in asupplemental treatment in addition to chemotherapy. Most chemotherapyagents used to target and destroy cancer cells act by killing allproliferating cells, i.e., cells going through cell division. Since bonemarrow is one of the most actively proliferating tissues in the body,hematopoietic stem cells are frequently damaged or destroyed bychemotherapy agents and in consequence, blood cell production isdiminishes or ceases. Chemotherapy must be terminated at intervals toallow the patient's hematopoietic system to replenish the blood cellsupply before resuming chemotherapy. It may take a month or more for theformerly quiescent stem cells to proliferate and increase the whiteblood cell count to acceptable levels so that chemotherapy may resume(when again, the bone marrow stem cells are destroyed).

While the blood cells regenerate between chemotherapy treatments,however, the cancer has time to grow and possibly become more resistantto the chemotherapy drugs due to natural selection. Therefore, thelonger chemotherapy is given and the shorter the duration betweentreatments, the greater the odds of successfully killing the cancer. Toshorten the time between chemotherapy treatments, embryonic-like stemcells or progenitor cells collected according to the methods of theinvention could be introduced into the patient. Such treatment wouldreduce the time the patient would exhibit a low blood cell count, andwould therefore permit earlier resumption of the chemotherapy treatment.

In another embodiment, the human placental stem cells can be used totreat or prevent genetic diseases such as chronic granulomatous disease.

5.6 Pharmaceutical Compositions

The present invention encompasses pharmaceutical compositions comprisinga dose and/or doses effective upon single or multiple administration,prior to or following transplantation of conditioned or unconditionedhuman progenitor stem cells, exerting effect sufficient to inhibit,modulate and/or regulate the differentiation of human pluripotent andmultipotent progenitor stem cells of placental origin into mesodermaland/or hematopoietic lineage cells.

In accordance with this embodiment, the embryonic-like stem cells of theinvention may be formulated as an injectable (e.g., PCT WO 96/39101,incorporated herein by reference in its entirety). In an alternativeembodiment, the cells and tissues of the present invention may beformulated using polymerizable or cross linking hydrogels as describedin U.S. Pat. Nos. 5,709,854; 5,516,532; 5,654,381; each of which isincorporated by reference in their entirety.

6. EXAMPLE 6.1. Example 1 Analysis of Cell Types Recovered fromPerfusate of Drained Placenta

This example describes the analysis of the cell types recovered from theeffluent perfusate of a placenta cultured according to the methods ofthe invention.

Twenty ml of phosphate buffered saline solution (PBS) was added to theperfusion liquid and a 10 ml portion was collected and centrifuged for25 minutes at 3000 rpm (revolutions per minute). The effluent wasdivided into four tubes and placed in an ice bath. 2.5 ml of a 1% fetalcalf serum (FCS) solution in PBS was added and the tubes werecentrifuged (140 minutes×10 g (acceleration due to gravity)). The pelletwas resuspended in 5 ml of 1% FCS and two tubes were combined. The totalmononucleocytes were calculated by adding the total lymphocytes and thetotal monocytes, and then multiplying the result by the total cellsuspension volume.

The following table discloses the types of cells obtained by perfusionof a cultured placenta according to the methods described hereinabove.

WBC Total # of 1000/ml Lym % MID % GRA % Volume Cells CB 10.5 43.2 848.8 60 ml 6.3 × 10⁸ (Cord Blood) PP 12.0 62.9 18.2 18.9 15 ml 1.8 × 10⁸(Placenta perfusate, room temperature) PP₂ 11.7 56.0 19.2 24.8 30 ml 3.5× 10⁸ (Placenta perfusate, 37° C.) Samples of PP were after Ficoll.Total cell number of PP after Ficoll was 5.3 × 10⁸ and number of CBbefore processing is 6.3 × 10⁸. Lym % indicates percent of lymphocytes;MID % indicates percent of midrange white blood cells; and GRA %indicates percent of granulocytes.

6.2. Example 2 Analysis of Cells Obtained by Perfusion and Incubation ofPlacenta

The following example describes an analysis of cells obtained byperfusion and incubation of placenta according to the methods of theinvention.

6.2.1. Materials and Methods

Placenta donors were recruited from expectant mothers that enrolled inprivate umbilical cord blood banking programs and provided informedconsent permitting the use of the exsanguinated placenta followingrecovery of cord blood for research purposes. Donor data may beconfidential. These donors also permitted use of blinded data generatedfrom the normal processing of their umbilical cord blood specimens forcryopreservation. This allowed comparison between the composition of thecollected cord blood and the effluent perfusate recovered using theexperimental method described below.

Following exsanguination of cord blood from the umbilical cord andplacenta is stored at room temperature and delivered to the laboratorywithin four to twenty-four hour, according to the methods describedhereinabove, the placenta was placed in a sterile, insulated containerat room temperature and delivered to the laboratory within 4 hours ofbirth. Placentas were discarded if, on inspection, they had evidence ofphysical damage such as fragmentation of the organ or avulsion ofumbilical vessels. Placentas were maintained at room temperature (23±2°C.) or refrigerated (4° C.) in sterile containers for 2 to 20 hours.Periodically, the placentas were immersed and washed in sterile salineat 25±3° C. to remove any visible surface blood or debris.

The umbilical cord was transected approximately 5 cm from its insertioninto the placenta and the umbilical vessels were cannulated with TEFLON®or polypropylene catheters connected to a sterile fluid path allowingbi-directional perfusion of the placenta and recovery of the effluentfluid. The methods described hereinabove enabled all aspects ofplacental conditioning, perfusion and effluent collection to beperformed under controlled ambient atmospheric conditions as well asreal-time monitoring of intravascular pressure and flow rates, core andperfusate temperatures and recovered effluent volumes. A range ofconditioning protocols were evaluated over a 24-hour postpartum period,and the cellular composition of the effluent fluid was analyzed by flowcytometry, light microscopy and colony forming unit assays.

6.2.2. Placental Conditioning

The donor placentas were processed at room temperature within 12 to 24hours after delivery. Before processing, the membranes were removed andthe maternal site washed clean of residual blood. The umbilical vesselswere cannulated with catheters made from 20 gauge Butterfly needles usefor blood sample collection.

The donor placentas were maintained under varying conditions such asmaintenance at 5-37° 5% CO₂, pH 7.2 to 7.5, preferably pH 7.45, in anattempt to simulate and sustain a physiologically compatible environmentfor the proliferation and recruitment of residual embryonic-like stemcells. The cannula was flushed with IMDM serum-free medium (GibcoBRL,NY) containing 2 U/ml heparin (Elkins-Sinn, NJ). Perfusion of theplacenta continued at a rate of 50 ml per minute until approximately 150ml of perfusate was collected. This volume of perfusate was labeled“early fraction.” Continued perfusion of the placenta at the same rateresulted in the collection of a second fraction of approximately 150 mland was labeled “late fraction.” During the course of the procedure, theplacenta was gently massaged to aid in the perfusion process and assistin the recovery of cellular material. Effluent fluid was collected fromthe perfusion circuit by both gravity drainage and aspiration throughthe arterial cannula.

Placentas were then perfused with heparinized (2 U/ml) Dulbecco'smodified Eagle Medium (H.DMEM) at the rate of 15 ml/minute for 10minutes and the perfusates were collected from the maternal sites withinone hour and the nucleated cells counted. The perfusion and collectionprocedures were repeated once or twice until the number of recoverednucleated cells fell below 100/ml. The perfusates were pooled andsubjected to light centrifugation to remove platelets, debris andde-nucleated cell membranes. The nucleated cells were then isolated byFicoll-Hypaque density gradient centrifugation and after washing,resuspended in H.DMEM. For isolation of the adherent cells, aliquots of5-10×10⁶ cells were placed in each of several T-75 flasks and culturedwith commercially available Mesenchymal Stem Cell Growth Medium (MSCGM)obtained from BioWhittaker, and placed in a tissue culture incubator(37° C., 5% CO₂). After 10 to 15 days, the non-adherent cells wereremoved by washing with PBS, which was then replaced by MSCGM. Theflasks were examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

6.2.3. Cell Recovery and Isolation

Cells were recovered from the perfusates by centrifugation at 5000×g for15 minutes at room temperature. This procedure served to separate cellsfrom contaminating debris and platelets. The cell pellets wereresuspended in IMDM serum-free medium containing 2 U/ml heparin and 2 mMEDTA (GibcoBRL, NY). The total mononuclear cell fraction was isolatedusing Lymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure and the mononuclear cell fractionwas resuspended. Cells were counted using a hemocytometer. Viability wasevaluated by trypan blue exclusion. Isolation of mesenchymal cells wasachieved by “differential trypsinization,” using a solution of 0.05%trypsin with 0.2% EDTA (Sigma, St. Louis Mo.). Differentialtrypsinization was possible because fibroblastoid cells detached fromplastic surfaces within about five minutes whereas the other adherentpopulations required more than 20-30 minutes incubation. The detachedfibroblastoid cells were harvested following trypsinization and trypsinneutralization, using Trypsin Neutralizing Solution (TNS, BioWhittaker).The cells were washed in H.DMEM and resuspended in MSCGM.

Flow cytometry was carried out using a Becton-Dickinson FACSCaliburinstrument and FITC and PE labeled monoclonal antibodies (mAbs),selected on the basis of known markers for bone marrow-derived MSC(mesenchymal stem cells), were purchased from B.D. and Caltaglaboratories (South San Francisco, Calif.), and SH2, SH3 and SH4antibody producing hybridomas were obtained from and reactivities of themAbs in their cultured supernatants were detected by FITC or PE labeledF(ab)′2 goat anti-mouse antibodies. Lineage differentiation was carriedout using commercially available induction and maintenance culture media(BioWhittaker), used as per manufacturer's instructions.

6.2.4. Isolation of Placental Embryonic-like Stem Cells

Microscopic examination of the adherent cells in the culture flasksrevealed morphologically different cell types. Spindle-shaped cells,round cells with large nuclei and numerous perinuclear small vacuoles,and star-shaped cells with several projections (through one of whichstar-shaped cells were attached to the flask) were observed adhering tothe culture flasks. Although no attempts were made to furthercharacterize these adherent cells, similar cells were observed in theculture of bone marrow, cord and peripheral blood, and thereforeconsidered to be non-stem cell-like in nature. The fibroblastoid cells,appearing last as clusters, were candidates for being MSC (mesenchymalstem cells) and were isolated by differential trypsinization andsubcultured in secondary flasks. Phase microscopy of the rounded cells,after trypsinization, revealed that the cells were highly granulated;indistinguishable from the bone marrow-derived MSC produced in thelaboratory or purchased from BioWhittaker. When subcultured, theplacenta-derived embryonic-like stem cells, in contrast to their earlierphase, adhered within hours, assumed characteristic fibroblastoid shape,and formed a growth pattern identical to the reference bonemarrow-derived MSC. During subculturing and refeeding, moreover, theloosely bound mononuclear cells were washed out and the culturesremained homogeneous and devoid of any visible non-fibroblastoid cellcontaminants.

6.2.5. Results

The expression of CD-34, CD-38, and other stem cell-associated surfacemarkers on early and late fraction purified mononuclear cells wasassessed by flow cytometry. Recovered, sorted cells were washed in PBSand then double-stained with antiCD34 phycoerythrin and anti-CD38fluorescein isothiocyanate (Becton Dickinson, Mountain View, Calif.).

Cell isolation was achieved by using magnetic cell separation, such asfor example, Auto Macs (Miltenyi). Preferably, CD 34+ cell isolation isperformed first.

6.3 Example 3 Perfusion Medium

The following example provides a formula of the preferred perfusatesolution for the cultivation of isolated placentas

Stock Final Chemical Source Concentration Concentration 500 ml DMEM-LGGibcoBRL11885- 300 ml 084 MCDB201 Sigma M-6770 dissolved in pH to 7.2.200 ml H2O filter FCS Hyclone 100% 2% 10 ml ITS Sigma I-3146 or 100x 1x5 ml GibcoBRL41400- 045 Pen&Strep GibcoBRL15140- 100x 1x 5 ml 122 LA +BSA Sigma + GibcoBRL 100x(1 μg/ml of 10 ng/ml of LA 5 ml BSA LADexamethasone Sigma D-2915 0.25 mM in H2O 0.05 μM 100 μl L-AscorbicSigma A-8960 1000x(100 mM) 1x(0.1 mM) 500 μl Acid PDGF (50 μg) R&D 220BD10 μg/ml in 10 ng/ml 500 μl 4 mM HCl + 0.1% BSA EGF (200 μg) SigmaE-9644 10 μg/ml in 10 ng/ml 500 μl 10 mM HAc + 0.1% BSA

The above-composition is a perfusate that may be used at a variety oftemperatures to perfuse placenta. It should be noted that additionalcomponents such as antibiotics, anticoagulant and other growth factorsmay be used in the perfusate or culture media.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

1. A method of treating an individual who has had a stroke, comprisingadministering to the individual a therapeutically effective amount ofisolated human placental stem cells, wherein said placental stem cellsare OCT-4⁺ and CD34⁻, wherein OCT-4 is octamer binding protein 4, andwherein said placental stem cell has at least the followingcharacteristics: CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH3⁺, SH4⁺,SSEA3⁻, and SSEA4⁻.
 2. A method of treating an individual who has had astroke, comprising administering to the individual therapeuticallyeffective amount of isolated human placental stem cells, wherein saidplacental stem cells are OCT-4⁺, CD34⁻, SSEA3⁻ and SSEA4⁻.
 3. A methodof treating an individual who has had a stroke, comprising administeringto the individual therapeutically effective amount of isolated humanplacental stem cells, wherein said placental stem cells are OCT-4⁺ andCD34⁻, and additionally SH3⁺ or SH4⁺.
 4. The method of claim 3, whereinsaid isolated placental stem cells have at least one of the followingcharacteristics: CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SSEA3⁻, orSSEA4⁻.
 5. The method of claim 3, wherein said isolated placental stemcells have at least the following characteristics: CD10⁺, CD29⁺, CD44⁺,CD45⁻, CD54⁺, CD90⁺, SSEA3⁻, and SSEA4⁻.
 6. The method of claim 3,wherein said isolated placental stem cells are SH3⁺ and SH4⁺.
 7. Themethod of claim 6, wherein said isolated human placental stem cells areSSEA3⁻ and SSEA4⁻.
 8. A method of treating an individual who hasischemia, comprising administering to the individual isolated tissueculture plastic-adherent human placental stem cells, wherein saidplacental stem cells are CD34⁻ and one or more of CD29⁺, CD45⁻, CD90⁺,SH2⁺, SH3⁺, SH4⁺, or MHC Class II⁻.
 9. The method of claim 8, whereinsaid isolated placental stem cells are CD34⁻, SH2⁺, SH3⁺ and SH4⁺. 10.The method of claim 8, wherein said isolated placental stem cells areCD34⁻ and MHC Class II⁻.
 11. The method of claim 8, wherein saidisolated placental stem cells are CD29⁺, CD34⁻, CD45⁻, CD90⁺, SH2⁺,SH3⁺, SH4⁺, and MHC Class II⁻.
 12. The method of claim 8, wherein saidischemia is a stroke.